Galectins regulate the inflammatory response in airway epithelial cells exposed to microbial neuraminidase by modulating the expression of SOCS1 and RIG1

Abstract

Influenza patients frequently display increased susceptibility to Streptococcus pneumoniae co-infection and sepsis, the prevalent cause of mortality during influenza pandemics. However, the detailed mechanisms by which an influenza infection predisposes patients to suffer pneumococcal pneumonia are not fully understood. A murine model for influenza infection closely reflects the observations in human patients, since if the animals that have recovered from influenza A virus (IAV) sublethal infection are challenged with S. pneumoniae, they undergo a usually fatal uncontrolled cytokine response. We have previously demonstrated both in vitro and in vivo that the expression and secretion of galectin-1 (Gal1) and galectin-3 (Gal3) are modulated during IAV infection, and that the viral neuraminidase unmasks galactosyl moieties in the airway epithelia. In this study we demonstrate in vitro that the binding of secreted Gal1 and Gal3 to the epithelial cell surface modulates the expression of SOCS1 and RIG1, and activation of ERK, AKT or JAK/STAT1 signaling pathways, leading to a disregulated expression and release of pro-inflammatory cytokines. Our results suggest that the activity of the viral and pneumococcal neuraminidases on the surface of the airway epithelial cells function as a "danger signal" that leads to rapid upregulation of SOCS1 expression to prevent an uncontrolled inflammatory response. The binding of extracellular Gal1 or Gal3 to the galactosyl moieties unmasked on the surface of airway epithelial cells can either "fine-tune" or severely disregulate this process, respectively, the latter potentially leading to hypercytokinemia.

Keywords: Chemokines; Cytokines; Galectin; Neuraminidase; RIG1; SOCS; Signaling pathway.

Fig 1. Exposure of lung carcinoma A549 cells to microbial neuraminidase and extracellular Gal1 and Gal3 modulates expression of SOCS1 and RIG1

Total RNA was extracted from A549 control (Ctrl) or neuraminidase (Arthrobacter ureafaciens and Clostridium perfringens) treated cells (NeuK) incubated in presence or absence of 15 μg/ml exogenous rhGal1 or rhGal3 for 1 h. (A) SOCS1 transcript level was analyzed by RT-PCR. (B) RIG1 transcript levels were analyzed by RT-PCR. Bar graphs show the fold change in mRNA expression levels in neuraminidase treated cells as well as galectin treated cells in comparison with control cell without neuraminidase and galectin treatment (Ctrl) after normalized to β-actin. All studies represent one of three independent experiments. *p <0.05; **p<0.001, non paired Student’s t test.

Fig 2. Activation of regulatory signaling pathways in A549 cells is modulated by exposure to microbial neuraminidase and extracellular Gal1 and Gal3

Total cell lysates were extracted from A549 control (Ctrl) or neuraminidase-treated cells (NeuK) incubated in the presence or absence of 15 μg/ml rhGal1 and rhGal3, and subject to 4 to 20% gradient SDS-PAGE. Total and phosphorylated protein levels of Integrin beta3 (INTb3), ERK, AKT, p38 MAPK, STAT1 and NF-κB were assessed by western blot. Bar graphs show the fold changes of phosphorylation levels in treated cells in comparison with control cells after normalization to total proteins. Shown images and the bar graphs are representative of data from at least two independent experiments. *p <0.05; **p<0.001, non paired Student’s t test.

Fig 3. Cytokines release by A549 cells

A549 control (Ctrl) or neuraminidase treated (NeuK) cells were stimulated in presence or absence of 15 μg/ml exogenous rhGal1 and rhGal3. The cytokines (IFN-γ, TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-12 and IL-15) released to the culture medium was measured by MSD multi-spot assay. *p <0.05; **p<0.001, non paired Student’s t test.